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This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Pipe, S. W. Articles by Kaufman, R. J. Search for Related Content PubMed PubMed Citation Articles by Pipe, S. W. Articles by Kaufman, R. J. Related Collections Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 February 2001, Vol. 97, No. 3, pp. 685-691 HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa Steven W. Pipe, Evgueni L. Saenko, Angela N. Eickhorst, Geoffrey Kemball-Cook, and Randal J. Kaufman From the Departments of Pediatrics and Biological Chemistry, Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, MI; Holland Laboratory, American Red Cross, Rockville, MD; and Haemostasis Research Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, London, United Kingdom. Thrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit (amino acid residues 373-740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. Patients with hemophilia A have been described whose plasmas display a discrepancy between their FVIII activities, where the 1-stage activity assay displays greater activity than the 2-stage activity assay. The molecular basis for one of these mutations, ARG531HIS, is an increased rate of A2 subunit dissociation. Examination of a homology model of the A domains of FVIII predicted ARG531 to lie at the interface of the A1 and A2 subunits and stabilize their interaction. Indeed, patients with mutations either directly contacting ARG531 (ALA284GLU, ALA284PRO) or closely adjacent to the A1-A2 interface in the tightly packed hydrophobic core (SER289LEU) have the same phenotype of 1-stage/2-stage discrepancy. The ALA284GLU and SER289LEU mutations in FVIII were produced by transfection of COS-1 monkey cells. Compared to FVIII wild-type both mutants had reduced specific activity by 1-stage clotting activity and at least a 2-fold lower activity by 2-stage analysis (COAMATIC), similar to the reported clinical data. Analysis of immunoaffinity purified ALA284GLU and SER289LEU proteins in an optical biosensor demonstrated that A2 dissociation was 3-fold faster for both FVIIIa mutants compared to FVIIIa wild-type. Therefore, these mutations within the A1 subunit of FVIIIa introduce a similar destabilization of the FVIIIa heterotrimer compared to the ARG531HIS mutation within the A2 subunit and support that these residues stabilize the A domain interface of FVIIIa. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: H. Wakabayashi and P. J. Fay Identification of Residues Contributing to A2 Domain-dependent Structural Stability in Factor VIII and Factor VIIIa J. Biol. Chem., April 25, 2008; 283(17): 11645 - 11651. [Abstract] [Full Text] [PDF] C. B. Doering, J. F. Healey, E. T. Parker, R. T. Barrow, and P. Lollar High Level Expression of Recombinant Porcine Coagulation Factor VIII J. Biol. Chem., October 4, 2002; 277(41): 38345 - 38349. [Abstract] [Full Text] [PDF]

	Copyright © 2001 by American Society of Hematology         Online ISSN: 1528-0020